Cancer is the second leading cause of death in the United States and US Veterans. Over 28% of cancer diagnoses in US Veterans in 2009 were for prostate, breast, and ovarian carcinoma. However, these cancers are often asymptomatic in beginning stages of the disease. Prostate cancer is a major health problem in male Veterans and it has been estimated that over 25% already have prostate cancer. Gender-associated breast and ovarian cancers are also of concern, especially since one out of every 15 Veterans is female. A VA Center for Women has been established in order to better address the rising medical needs of our female Veterans. While screening and detection methods exist for these cancers, they all have limitations. It can be envisioned that the use of molecules that specifically target antigens on carcinomas and metastases will lead to improved cancer detection and treatment modalities. Radiolabeled antibodies and peptides are currently being explored as diagnostic cancer imaging agents. However, high molecular weight antibodies as well as small peptides may not be ideal as cancer imaging agents in vivo. Only a small number of radiolabeled antibodies and peptides possess the requisite high affinity, specificity, stability, and tumor uptake to serve as cancer imaging agents. Thus, finding new cancer targeting vehicles and cancer-associated antigens is a central goal of the field. Development of specific tumor-targeting molecules that can be used safely and non- invasively to detect and treat cancer is undeniably an important priority for the Veterans Administration if it is to meet the health care challenges of our past, current, and future military personnel. New peptide-based molecular probes to facilitate cancer imaging are rapidly evolving due to implementation of bacteriophage (phage) display approaches. While radiolabeled peptides have shown good tumor-targeting propensity in vitro, their translation into the clinic has been slowed by sub-optimal tumor retention and almost universal high renal uptake and retention. We have used phage display to obtain peptides that target the tumor-associated ErbB-2 receptor, galectin-3 (gal-3) and its carbohydrate ligand Thomsen- Friedenreich (TF) antigen, which are involved in cancer cell adhesion and signaling. We hypothesize that peptides that bind ErbB-2, gal-3, and TF grafted into constrained loops of polypeptide scaffolds once radiolabeled, will form the foundation for novel single photon emission computed tomography (SPECT) and positron emission computed tomography (PET) imaging agents for prostate, breast, and ovarian tumors. We will expand the applications of phage display to employ innovative functional selection approaches in vivo to improve the imaging and, potentially, therapeutic efficacy of the tumor-targeting radiolabeled peptides. The radiolabeled conjugates may serve as diagnostic radiopharmaceuticals for the detection of primary and metastatic cancer and indicators of response to therapy. In the long term, the peptides may function in vivo to reduce tumor growth and metastasis by blocking signaling pathways involved in tumorigenesis. Diverse molecular biology, protein engineering, radiochemistry, and optical imaging approaches will be employed. The objectives of this proposed research are to: 1) select phage display libraries based on the ErbB-2 and gal-3-targeting sequences in mice in order to identify phage and corresponding peptides with high tumor uptake and low kidney retention; 2) engineer the optimized peptide motifs into stable small molecular weight cysteine-constrained scaffolds for enhanced in vivo stability, affinity, and rapid excretion; 3) compare the in vivo SPECT imaging efficacy of 111Indium-radiolabeled ErbB-2-, gal-3-, and TF- targeting linear peptides and engineered peptide scaffolds in at least one appropriate tumor model (i.e. breast, prostate or ovary); and 4) develop 64Copper-labeled peptide counterparts for sensitive PET imaging. This work is significant and relevant to the VA mission because the peptides discovered here may translate into novel prostate, breast, and ovarian cancer diagnostics and therapeutics for our veterans.